Journal: Cell metabolism

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells

doi: 10.1016/j.cmet.2018.10.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The human breast cancer MCF7 and MDA-MB-231 cells, human cervical cancer HeLa cells, human osteosarcoma 143B cells, mouse mastocytoma P815 cells and human pancreatic cancer PANC-1 cells (all from the ATCC), as well as human pancreatic cancer PaTu8902 cells (DMSZ) were maintained in the same media as B16 cells without uridine and pyruvate. .

Techniques: Glo Assay, Pyruvate Assay, Bicinchoninic Acid Protein Assay, Labeling, Recombinant, Microarray, Cloning, Binding Assay, Software, Imaging, Protease Inhibitor, DNA Extraction, SYBR Green Assay

Journal: Cancers

Article Title: Targeting CSF1R Alone or in Combination with PD1 in Experimental Glioma

doi: 10.3390/cancers13102400

Figure Lengend Snippet: CSF1R and PD1 are present in primary and progressive glioblastoma. Representative tumor areas from matched pairs of newly diagnosed and progressive glioblastoma. H&E staining (top row) and immunohistochemical staining of CSF1R ( n = 28), CD204 ( n = 27), CD163 ( n = 31), PD1 ( n = 30), PD-L1 ( n = 31), CD3 ( n = 28), CD4 ( n = 30), and CD8 ( n = 28). Scale bars 50 µm.

Article Snippet: Formalin-fixed, paraffin-embedded tissue microarray sections were stained for CD3 (1:500, 40 min CC1 pretreatment, clone SP7, ThermoFisher, Waltham, MA, USA), CD4 (1:2, 24 min CC1, clone SP35, Ventana Medical Systems, Roche Group, Indianapolis, IN, USA), CD8 (RTU, 64 min CC1, clone SP57, Ventana Medical Systems, Roche Group, USA), CD163 (RTU, MRQ-26, Ventana Medical Systems, Roche Group, USA), CSF1R (dilution 1:2500, 32 min CC1, clone 29, Roche Diagnostics GmbH, Penzberg, Germany), PD1 (1:100, 64 min CC2, Clone MRQ-22, Zytomed, Berlin, Germany), PD-L1 (1:100, 64 min CC1, ab205921, Abcam, Cambridge, UK), and CD204 (1:2500, 32 min CC1, HPA000272, Sigma Aldrich, St. Louis, MI, USA) on the Ventana Benchmark XT. immunohistochemistry system with a 32 min antibody incubation time each.

Techniques: Staining, Immunohistochemical staining

Journal: PLoS Genetics

Article Title: Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus

doi: 10.1371/journal.pgen.1007755

Figure Lengend Snippet: LocusZoom plot depicting association results at rs2820315 locus at chromosome 1q32.1 in (A) CAD from CARDIoGRAMplusC4D + UKB GWAS, (B) LDL cholesterol from Global Lipid Genetics Consortium and rs2820315 eQTL association (highlighting LD SNP rs34091558) with LMOD1 and other candidate genes in (C) Tibial Artery and (D) Liver in the GTEx dataset. Circles represent SNPs associated using an additive or recessive model, color-coded for LD (r 2 ) with the lead GWAS SNP rs2820315 (purple diamond), which resides near the LMOD1 gene.

Article Snippet: For detecting LMOD1 expression within the atherosclerotic lesion, brachiocephalic artery tissue sections were stained with a primary antibody specific to LMOD1 (Proteintech, 1:400) as previously described[ ].

Techniques:

Journal: PLoS Genetics

Article Title: Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus

doi: 10.1371/journal.pgen.1007755

Figure Lengend Snippet: (A) Schematic indicating neighboring genes in the LMOD1 locus using RefSeq database, coding transcripts in blue and non-coding transcripts in green (upper panel). Expression profile of coding and non-coding transcripts at LMOD1 locus in coronary artery tissues (n = 133) from GTEx database (v6p) (lower panel). (B) Tissue expression profile of LMOD1 and SHISA4 across the entire GTEx dataset (v7) ranked according to median transcripts per million (TPM) for each tissue. Boxplots showing rs2820315 allele dosage correlated with LMOD1 (C) and rs34091558 allele dosage correlated with LMOD1 (D) in tibial artery tissue in GTEx dataset (v7). (E) Allelic expression imbalance (AEI) for LMOD1 in HCASMCs heterozygous at rs2820315 and rs34091558, using rs2820312 coding SNP as a proxy (n = 12). Values represent mean ± standard deviation of triplicates of cDNA/gRNA normalized allelic ratios.

Article Snippet: For detecting LMOD1 expression within the atherosclerotic lesion, brachiocephalic artery tissue sections were stained with a primary antibody specific to LMOD1 (Proteintech, 1:400) as previously described[ ].

Techniques: Expressing, Standard Deviation

Journal: PLoS Genetics

Article Title: Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus

doi: 10.1371/journal.pgen.1007755

Figure Lengend Snippet: (A) Prediction of rs34091558 non-risk (TA) and risk allele (T) on FOXO3 binding based on JASPAR PWM scores. Human sequence (hg38) is shown aligned to the consensus FOXO3 sequence and mammalian genomic sequences and PhyloP conservation track. (B) Relative expression (RPKM) of candidate Forkhead transcription factors in coronary artery tissues (n = 133) from the GTEx dataset. (C) Luciferase activity examined for each of the LMOD1 enhancer constructs in A7r5 SMCs in the presence of FOXO3 . Results were reproduced in n = 3 independent experiments performed in quadruplicates. (D) Chromatin immunoprecipitation (ChIP) assay for LMOD1 and EGFR (as a positive control region) in HCASMC chromatin lysates immunoprecipitated with antibodies to FOXO3 or a negative control rabbit IgG. Results were repeated in n = 3 independent studies. (E) Allele specific ChIP (haploChIP) for FOXO3 protein in DNA derived from cultured HCASMC ChIP experiments. DNA from cell line homozygous for the ancestral allele was used as a positive control and arbitrarily set to 1. Values represent mean ± standard deviation of triplicates. Similar results were observed from n = 4 independent lines for each genotype. (F) Quantitative RT-PCR analysis of LMOD1 in HCASMCs following knock down of endogenous FOXO3 (F) or overexpression of FOXO3 (G). The results were reproduced in n = 3 independent experiments.

Article Snippet: For detecting LMOD1 expression within the atherosclerotic lesion, brachiocephalic artery tissue sections were stained with a primary antibody specific to LMOD1 (Proteintech, 1:400) as previously described[ ].

Techniques: Binding Assay, Sequencing, Genomic Sequencing, Expressing, Luciferase, Activity Assay, Construct, Chromatin Immunoprecipitation, Positive Control, Immunoprecipitation, Negative Control, Derivative Assay, Cell Culture, Standard Deviation, Quantitative RT-PCR, Knockdown, Over Expression

Journal: PLoS Genetics

Article Title: Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus

doi: 10.1371/journal.pgen.1007755

Figure Lengend Snippet: (A) UCSC Browser screenshot of the LMOD1 CAD locus at chromosome 1q32.1 highlighting the candidate causal variant, rs34091558, overlapping ATAC-seq open chromatin and RNA-seq tracks in HCASMCs treated with PDGF-BB (n = 2 biological replicates). Genomic coordinates refer to hg19 assembly. Quantitative RT-PCR (B) and Western blotting (C) data revealing PDGF-BB and phosphorylated-FOXO3 (P-FOXO3) mediated LMOD1 expression. (D) Co-expression microarray analysis performed in a cohort of carotid atherosclerotic plaques (n = 127) indicating that LMOD1 and FOXO3 are positively correlated in arterial tissues.

Article Snippet: For detecting LMOD1 expression within the atherosclerotic lesion, brachiocephalic artery tissue sections were stained with a primary antibody specific to LMOD1 (Proteintech, 1:400) as previously described[ ].

Techniques: Variant Assay, RNA Sequencing, Quantitative RT-PCR, Western Blot, Expressing, Microarray

Journal: PLoS Genetics

Article Title: Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus

doi: 10.1371/journal.pgen.1007755

Figure Lengend Snippet: (A) Quantitative RT-PCR and Western blotting analysis confirming endogenous knockdown of LMOD1 in cultured HCASMCs. Cell proliferation measured in HCASMCs transfected with siCtrl or si LMOD1 via (B) trypan blue exclusion and (C) CellTiter96 Non-Radioactive Cell Proliferation Assay (MTT assay). (D) Differences in cell migration assessed via Boyden Chamber assay in HCASMCs transfected with siCtrl or si LMOD1 . (E) Images of cell contraction in siCtrl or si LMOD1 transfected HCASMCs at indicated time points and represented via quantification (F-G). All data represent mean ± standard deviation from n = 3 independent experiments.

Article Snippet: For detecting LMOD1 expression within the atherosclerotic lesion, brachiocephalic artery tissue sections were stained with a primary antibody specific to LMOD1 (Proteintech, 1:400) as previously described[ ].

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Cell Culture, Transfection, Proliferation Assay, MTT Assay, Migration, Boyden Chamber Assay, Standard Deviation

Journal: PLoS Genetics

Article Title: Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus

doi: 10.1371/journal.pgen.1007755

Figure Lengend Snippet: (A and D) Immunofluorescence staining for LMOD1, Tomato and DAPI in the brachiocephalic artery of 24 week old Myh11 - Cre-ER T2 ROSA26-STOP -tdTomato Apoe -/- mice fed high fat diet for 18 weeks. (B and C) LMOD1 and tomato expressing cells were identified in the fibrous cap (white arrows). (E and F) Another lesion showing LMOD1 and tomato expressing cells in the media (blue arrows). Tomato positive cells in the lesion stained negative for LMOD1 in the lesion. Images were captured using a 20x objective and are representative of n = 3 independent mice.

Article Snippet: For detecting LMOD1 expression within the atherosclerotic lesion, brachiocephalic artery tissue sections were stained with a primary antibody specific to LMOD1 (Proteintech, 1:400) as previously described[ ].

Techniques: Immunofluorescence, Staining, Expressing

Journal: PLoS Genetics

Article Title: Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus

doi: 10.1371/journal.pgen.1007755

Figure Lengend Snippet: Individuals having the rs34091558-TA ancestral, protective allele would be at reduced CAD risk due to greater LMOD1 expression levels, whereas individuals having the rs34091558-T derived, risk allele would be at greater CAD risk due to reduced LMOD1 expression levels, through a FOXO3-dependent mechanism.

Article Snippet: For detecting LMOD1 expression within the atherosclerotic lesion, brachiocephalic artery tissue sections were stained with a primary antibody specific to LMOD1 (Proteintech, 1:400) as previously described[ ].

Techniques: Expressing, Derivative Assay

Journal: Biosensors & bioelectronics

Article Title: Microarray method to monitor 40 intestinal bacterial species in the study of azo dye reduction

doi: 10.1016/j.bios.2004.04.011

Figure Lengend Snippet: Bacteria and the probe numbers in the microarray

Article Snippet: Anaerobic bacteria were cultured at 35 °C in either prereduced anaerobically sterilized (PRAS) Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin (Remel, Lenexa, KS, USA), inoculated under an oxygen-free cannula using 85% nitrogen, 10% hydrogen and 5% carbon dioxide, or on PRAS brucella blood agar plates supplemented with vitamin K and hemin (Remel). table ft1 table-wrap mode="anchored" t5 caption a7 Number Bacterial species and strain Probe number 1 B. thetaiotaomicron ATCC 29148 1, 2, 3 2 B. vulgatus ATCC 8482 4, 5, 6 3 B. fragilis ATCC 23745 7, 8, 9 4 B. distasonis ATCC 8503 10, 11, 12 5 C. clostridioforme ATCC 29084 13, 14, 15 6 C. leptum ATCC 29065 16, 17, 18 7 F. prausnitzii ATCC 27768 19, 20, 21 8 P. productus ATCC 27340 22, 23, 24 9 R. obeum ATCC 29174 25, 26, 27 10 R. bromii ATCC 27255 28, 29, 30 11 R. callidus ATCC 27760 31, 32, 33 12 R. albus ATCC 27210 34, 35, 36 13 B. longum ATCC 15707 37, 38, 39 14 B. adolescentis ATCC 15703 40, 41, 42 15 B. infantis ATCC 15697 43, 44, 45 16 E. biforme ATCC 27806 46, 47, 48 17 E. aerofaciens ATCC 25986 49, 50, 51 18 L. acidophilus ATCC 4356 52, 53, 54 19 E. coli ATCC 25922 55, 56, 57 20 E. faecium ATCC 19434 58, 59, 60 21 B. uniformis ATCC 8492 61, 62, 63 22 B. ovatus ATCC 8483 64, 65, 66 23 B. caccae ATCC 43185 67, 68, 69 24 C. perfringens ATCC 13124 70, 71, 72 25 C. butyricum ATCC 19398 73, 74, 75 26 C. ramosum ATCC 25582 76, 77, 78 27 C. difficile ATCC 9689 79, 80, 81 28 C. indolis ATCC 25771 82, 83, 84 29 F. russii ATCC 25533 85, 86, 87 30 F. nucleatum ATCC 25586 88, 89, 90 31 B. catenulatum ATCC 27539 91, 92, 93 32 B. angulatum ATCC 27535 94, 95, 96 33 E. rectale ATCC 33656 97, 98, 99 34 E. eligens ATCC 27750 100, 101, 102 35 E. limosum ATCC 8486 103, 104, 105 36 E. lentum ATCC 25553 106, 107, 108 37 L. fermentum ATCC 9338 109, 110, 111 38 E. faecalis ATCC 27274 112, 113, 114 39 P. magnus ATCC 14955 115, 116, 117 40 R. gnavus ATCC 291492 118, 119, 120 Open in a separate window Bacteria and the probe numbers in the microarray

Techniques: Bacteria

Journal: Biosensors & bioelectronics

Article Title: Microarray method to monitor 40 intestinal bacterial species in the study of azo dye reduction

doi: 10.1016/j.bios.2004.04.011

Figure Lengend Snippet: Microarray test results read from

Article Snippet: Anaerobic bacteria were cultured at 35 °C in either prereduced anaerobically sterilized (PRAS) Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin (Remel, Lenexa, KS, USA), inoculated under an oxygen-free cannula using 85% nitrogen, 10% hydrogen and 5% carbon dioxide, or on PRAS brucella blood agar plates supplemented with vitamin K and hemin (Remel). table ft1 table-wrap mode="anchored" t5 caption a7 Number Bacterial species and strain Probe number 1 B. thetaiotaomicron ATCC 29148 1, 2, 3 2 B. vulgatus ATCC 8482 4, 5, 6 3 B. fragilis ATCC 23745 7, 8, 9 4 B. distasonis ATCC 8503 10, 11, 12 5 C. clostridioforme ATCC 29084 13, 14, 15 6 C. leptum ATCC 29065 16, 17, 18 7 F. prausnitzii ATCC 27768 19, 20, 21 8 P. productus ATCC 27340 22, 23, 24 9 R. obeum ATCC 29174 25, 26, 27 10 R. bromii ATCC 27255 28, 29, 30 11 R. callidus ATCC 27760 31, 32, 33 12 R. albus ATCC 27210 34, 35, 36 13 B. longum ATCC 15707 37, 38, 39 14 B. adolescentis ATCC 15703 40, 41, 42 15 B. infantis ATCC 15697 43, 44, 45 16 E. biforme ATCC 27806 46, 47, 48 17 E. aerofaciens ATCC 25986 49, 50, 51 18 L. acidophilus ATCC 4356 52, 53, 54 19 E. coli ATCC 25922 55, 56, 57 20 E. faecium ATCC 19434 58, 59, 60 21 B. uniformis ATCC 8492 61, 62, 63 22 B. ovatus ATCC 8483 64, 65, 66 23 B. caccae ATCC 43185 67, 68, 69 24 C. perfringens ATCC 13124 70, 71, 72 25 C. butyricum ATCC 19398 73, 74, 75 26 C. ramosum ATCC 25582 76, 77, 78 27 C. difficile ATCC 9689 79, 80, 81 28 C. indolis ATCC 25771 82, 83, 84 29 F. russii ATCC 25533 85, 86, 87 30 F. nucleatum ATCC 25586 88, 89, 90 31 B. catenulatum ATCC 27539 91, 92, 93 32 B. angulatum ATCC 27535 94, 95, 96 33 E. rectale ATCC 33656 97, 98, 99 34 E. eligens ATCC 27750 100, 101, 102 35 E. limosum ATCC 8486 103, 104, 105 36 E. lentum ATCC 25553 106, 107, 108 37 L. fermentum ATCC 9338 109, 110, 111 38 E. faecalis ATCC 27274 112, 113, 114 39 P. magnus ATCC 14955 115, 116, 117 40 R. gnavus ATCC 291492 118, 119, 120 Open in a separate window Bacteria and the probe numbers in the microarray

Techniques: Microarray

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dihydropyrimidinase Like 2 Promotes Bladder Cancer Progression via Pyruvate Kinase M2-Induced Aerobic Glycolysis and Epithelial–Mesenchymal Transition

doi: 10.3389/fcell.2021.641432

Figure Lengend Snippet: Dihydropyrimidinase like 2 (DPYSL2) upregulation correlated with tumor staging and poor survival in patients with bladder cancer. (A,B) In the GSE89 and GSE32548 datasets, DPYSL2 mRNA levels were significantly higher in muscular invasive bladder cancer (MIBC) samples (≥T2) than those in non-muscular invasive bladder cancer (NMIBC) samples (Ta and T1). (C–E) DPYSL2 mRNA levels were significantly increased in infiltrating bladder urothelial carcinoma compared with those in superficial bladder cancer in the Oncomine database. (F–H) In a TCGA bladder cancer specimen cohort ( n = 406), the patients with high DPYSL2 expression had increased death rates (F) and decreased overall survival (G) and recurrence-free survival (H) , compared with the patients with low DPYSL2 expression. (I,J) Immunohistochemical staining was performed to detect the protein levels of DPYSL2 in a bladder cancer tissue microarray containing non-cancerous ( n = 16), NMIBC ( n = 48), and MIBC ( n = 128) tissue samples. Representative images are shown. Magnification 40×, 200×. (K,L) Quantitative real-time PCR (qRT-PCR) and Western blot analysis were conducted to measure mRNA and protein levels of DPYSL2 in randomly selected paired fresh-frozen bladder cancer and adjacent non-cancerous bladder tissue samples. Actin was used as an internal reference. Data are expressed as the mean ± standard error of the mean (SEM). * P < 0.05, ** P < 0.01, *** P < 0.001; n = 6. T, tumor; TCGA, The Cancer Genome Atlas; DPYSL2, dihydropyrimidinase like 2; NMIBC, non-muscular invasive bladder cancer; MIBC, muscular invasive bladder cancer; NT, non-cancerous tissue.

Article Snippet: After incubated with 5% bovine serum albumin at room temperature for 2 h, The membrane was incubated with primary antibody against DPYSL2 (1:1000; 10,188-1-AP; Proteintech), Flag (1:1000; ab1162; Abcam, Cambridge, United Kingdom), PKM1 (1:2000; 15,821-1-AP; Proteintech), PKM2 (1:2000; 15,822-1-AP; Proteintech), E-cadherin (1:2000; #14,472; CST, Danvers, MA, United States), Vimentin (1:2000; #5741; CST), ZEB (1:1000; #9585; CST), or β-actin (1:4000; sc-81178; Santa Cruz Biotechnology, Dallas, TX, United States) overnight at 4°C.

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dihydropyrimidinase Like 2 Promotes Bladder Cancer Progression via Pyruvate Kinase M2-Induced Aerobic Glycolysis and Epithelial–Mesenchymal Transition

doi: 10.3389/fcell.2021.641432

Figure Lengend Snippet: Comparison of clinical features between bladder cancer patients with low and high # DPYSL2 mRNA levels in the TCGA database.

Article Snippet: After incubated with 5% bovine serum albumin at room temperature for 2 h, The membrane was incubated with primary antibody against DPYSL2 (1:1000; 10,188-1-AP; Proteintech), Flag (1:1000; ab1162; Abcam, Cambridge, United Kingdom), PKM1 (1:2000; 15,821-1-AP; Proteintech), PKM2 (1:2000; 15,822-1-AP; Proteintech), E-cadherin (1:2000; #14,472; CST, Danvers, MA, United States), Vimentin (1:2000; #5741; CST), ZEB (1:1000; #9585; CST), or β-actin (1:4000; sc-81178; Santa Cruz Biotechnology, Dallas, TX, United States) overnight at 4°C.

Techniques: Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dihydropyrimidinase Like 2 Promotes Bladder Cancer Progression via Pyruvate Kinase M2-Induced Aerobic Glycolysis and Epithelial–Mesenchymal Transition

doi: 10.3389/fcell.2021.641432

Figure Lengend Snippet: Knockdown of DPYSL2 inhibited cell proliferation, colony formation, migration, and invasion of bladder cancer cells. (A) T24 cells were transfected with small interfering RNA against DPYSL2 (siDPYSL2) or negative control. DPYSL2 was immunostained with an anti-DPYSL2 antibody and detected by immunofluorescence microscopy. Representative images are shown. Magnification 200×. (B,C) 5637 and T24 cells were transfected with siDPYSL2 or negative control. qRT-PCR and Western blot analysis were performed to measure the mRNA and protein levels of DPYSL2. (D–F) Cell proliferation, colony formation, migration, and invasion assays were conducted. Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001; n = 3. NC, negative control; si, small interfering RNA.

Article Snippet: After incubated with 5% bovine serum albumin at room temperature for 2 h, The membrane was incubated with primary antibody against DPYSL2 (1:1000; 10,188-1-AP; Proteintech), Flag (1:1000; ab1162; Abcam, Cambridge, United Kingdom), PKM1 (1:2000; 15,821-1-AP; Proteintech), PKM2 (1:2000; 15,822-1-AP; Proteintech), E-cadherin (1:2000; #14,472; CST, Danvers, MA, United States), Vimentin (1:2000; #5741; CST), ZEB (1:1000; #9585; CST), or β-actin (1:4000; sc-81178; Santa Cruz Biotechnology, Dallas, TX, United States) overnight at 4°C.

Techniques: Knockdown, Migration, Transfection, Small Interfering RNA, Negative Control, Immunofluorescence, Microscopy, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dihydropyrimidinase Like 2 Promotes Bladder Cancer Progression via Pyruvate Kinase M2-Induced Aerobic Glycolysis and Epithelial–Mesenchymal Transition

doi: 10.3389/fcell.2021.641432

Figure Lengend Snippet: Dihydropyrimidinase like 2 (DPYSL2) overexpression promoted bladder cancer cell proliferation, colony formation, migration, and invasion in vitro , as well as tumor growth and metastasis in vivo . (A,B) T24 cells were transfected with the Flag-DPYSL2 plasmids or negative control. Flag and DPYSL2 were immunostained with anti-Flag (A) or anti-DPYSL2 (B) antibody. Representative images are shown. Magnification 200×. (C,D) 5637 and T24 cells were stably transfected with empty lentiviral vectors (LV-control) or lentiviral vectors overexpressing DPYSL2 (LV-DPYSL2). qRT-PCR and Western blot analysis were conducted to measure the mRNA and protein levels of DPYSL2. (E–G) Cell proliferation, colony formation, migration, and invasion assays were conducted. (H) NOD-SCID mice were subcutaneously inoculated with 4 × 10 6 LV-control- or LV-DPYSL2-transfected 5637 cells at the left and right armpits ( n = 5/group). Mice were euthanized at 4 weeks after inoculation, and the tumors were collected, measured, and weighed. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; n = 5. (I) NOD-SCID mice were injected with 2 × 10 6 LV-control- or LV-DPYSL2-transfected 5637 cells through caudal vein ( n = 6/group). At 3 months after injection, each mouse was injected with 0.15 mg/g potassium d-fluorescein through the tail vein. The IVIS 200 imaging system was used to detect luminescence in tumor-bearing mice. (J) Upper panel: Representative images of pulmonary metastases. Lower panel: Hematoxylin and eosin staining of pulmonary metastases. Red arrows indicate metastases.

Article Snippet: After incubated with 5% bovine serum albumin at room temperature for 2 h, The membrane was incubated with primary antibody against DPYSL2 (1:1000; 10,188-1-AP; Proteintech), Flag (1:1000; ab1162; Abcam, Cambridge, United Kingdom), PKM1 (1:2000; 15,821-1-AP; Proteintech), PKM2 (1:2000; 15,822-1-AP; Proteintech), E-cadherin (1:2000; #14,472; CST, Danvers, MA, United States), Vimentin (1:2000; #5741; CST), ZEB (1:1000; #9585; CST), or β-actin (1:4000; sc-81178; Santa Cruz Biotechnology, Dallas, TX, United States) overnight at 4°C.

Techniques: Over Expression, Migration, In Vitro, In Vivo, Transfection, Negative Control, Stable Transfection, Control, Quantitative RT-PCR, Western Blot, Injection, Imaging, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dihydropyrimidinase Like 2 Promotes Bladder Cancer Progression via Pyruvate Kinase M2-Induced Aerobic Glycolysis and Epithelial–Mesenchymal Transition

doi: 10.3389/fcell.2021.641432

Figure Lengend Snippet: Dihydropyrimidinase like 2 (DPYSL2) interacted with pyruvate kinase M2 (PKM2) in bladder cancer cells. (A) Co-immunoprecipitation (Co-IP), followed by silver staining and mass spectrometry, was performed to identify the proteins that physically interacted with DPYSL2. (B) A protein-protein interaction network was established to predict the interactions of DPYSL2 with potential candidate proteins using the STRING database ( https://string-db.org/ ). (C) HEK293T cells were transfected with Flag or Flag-DPYSL2 plasmids. The Flag-DPYSL2 complexes were co-immunoprecipitated using an anti-Flag antibody, followed by Western blot analysis to detect PKM1 and PKM2. (D) HEK293T cells were transfected with HA or HA-PKM2 plasmids. The HA-PKM2 complexes were co-immunoprecipitated using an anti-HA antibody, followed by Western blot analysis to detect DPYSL2. (E,F) T24 cells were transfected with LV-DPYSL2, siDPYSL2, or corresponding negative control. A cross-link analysis was performed to detect the monomers, dimers, and tetramers of PKM2.

Article Snippet: After incubated with 5% bovine serum albumin at room temperature for 2 h, The membrane was incubated with primary antibody against DPYSL2 (1:1000; 10,188-1-AP; Proteintech), Flag (1:1000; ab1162; Abcam, Cambridge, United Kingdom), PKM1 (1:2000; 15,821-1-AP; Proteintech), PKM2 (1:2000; 15,822-1-AP; Proteintech), E-cadherin (1:2000; #14,472; CST, Danvers, MA, United States), Vimentin (1:2000; #5741; CST), ZEB (1:1000; #9585; CST), or β-actin (1:4000; sc-81178; Santa Cruz Biotechnology, Dallas, TX, United States) overnight at 4°C.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Silver Staining, Mass Spectrometry, Transfection, Western Blot, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dihydropyrimidinase Like 2 Promotes Bladder Cancer Progression via Pyruvate Kinase M2-Induced Aerobic Glycolysis and Epithelial–Mesenchymal Transition

doi: 10.3389/fcell.2021.641432

Figure Lengend Snippet: Pyruvate kinase M2 (PKM2) silencing completely abolished DPYSL2-enhanced malignant behaviors of bladder cancer cells. Stable Flag-DPYSL2-overexpressing 5637 or T24 cells were transfected with siPKM2 or negative control. The protein levels of Flag-DPYSL2 and PKM2 (A) , cell proliferation (B) , colony formation (C) , migration, and invasion (D) were examined. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, non-significant; n = 3.

Article Snippet: After incubated with 5% bovine serum albumin at room temperature for 2 h, The membrane was incubated with primary antibody against DPYSL2 (1:1000; 10,188-1-AP; Proteintech), Flag (1:1000; ab1162; Abcam, Cambridge, United Kingdom), PKM1 (1:2000; 15,821-1-AP; Proteintech), PKM2 (1:2000; 15,822-1-AP; Proteintech), E-cadherin (1:2000; #14,472; CST, Danvers, MA, United States), Vimentin (1:2000; #5741; CST), ZEB (1:1000; #9585; CST), or β-actin (1:4000; sc-81178; Santa Cruz Biotechnology, Dallas, TX, United States) overnight at 4°C.

Techniques: Transfection, Negative Control, Migration

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dihydropyrimidinase Like 2 Promotes Bladder Cancer Progression via Pyruvate Kinase M2-Induced Aerobic Glycolysis and Epithelial–Mesenchymal Transition

doi: 10.3389/fcell.2021.641432

Figure Lengend Snippet: Dihydropyrimidinase like 2 (DPYSL2) promoted aerobic glycolysis and epithelial–mesenchymal transition (EMT) via PKM2. (A,B) Glucose uptake (A) and lactate production (B) were measured in 5637 and T24 cell lines stably overexpressing DPYSL2. (C,D) 5637 or T24 cells were transfected with Flag-DPYSL2 plasmids and siPKM2 individually or in combination. Glucose uptake (C) and lactate production (D) were measured, respectively, at 36 or 48 h after transfection. (E,F) Western blot analysis was performed to determine EMT marker expression in 5637 and T24 cell lines stably overexpressing DPYSL2 (E) and in three pairs of mouse tumors (F) . (G) 5637 or T24 cells Flag-DPYSL2 plasmids and siPKM2 individually or in combination. Western blot analysis was performed to measure EMT marker expression. Actin was used as an internal reference. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01; ns, non-significant; n = 3.

Article Snippet: After incubated with 5% bovine serum albumin at room temperature for 2 h, The membrane was incubated with primary antibody against DPYSL2 (1:1000; 10,188-1-AP; Proteintech), Flag (1:1000; ab1162; Abcam, Cambridge, United Kingdom), PKM1 (1:2000; 15,821-1-AP; Proteintech), PKM2 (1:2000; 15,822-1-AP; Proteintech), E-cadherin (1:2000; #14,472; CST, Danvers, MA, United States), Vimentin (1:2000; #5741; CST), ZEB (1:1000; #9585; CST), or β-actin (1:4000; sc-81178; Santa Cruz Biotechnology, Dallas, TX, United States) overnight at 4°C.

Techniques: Stable Transfection, Transfection, Western Blot, Marker, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dihydropyrimidinase Like 2 Promotes Bladder Cancer Progression via Pyruvate Kinase M2-Induced Aerobic Glycolysis and Epithelial–Mesenchymal Transition

doi: 10.3389/fcell.2021.641432

Figure Lengend Snippet: Glycolysis inhibitor dichloroacetate (DCA) abolished DPYSL2-induced aerobic glycolysis and EMT marker alterations in bladder cancer cells. (A–C) 5637 or T24 cells were transfected with Flag-DPYSL2 and incubated for 12 h, followed by DCA (100 μM) treatment for 36 h. Glucose uptake (A) , lactate production (B) , and protein levels of EMT markers (C) were measured. (D) A schematic diagram illustrates that DPYSL2 promotes EMT and aerobic glycolysis by interacting with PKM2. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, non-significant.

Article Snippet: After incubated with 5% bovine serum albumin at room temperature for 2 h, The membrane was incubated with primary antibody against DPYSL2 (1:1000; 10,188-1-AP; Proteintech), Flag (1:1000; ab1162; Abcam, Cambridge, United Kingdom), PKM1 (1:2000; 15,821-1-AP; Proteintech), PKM2 (1:2000; 15,822-1-AP; Proteintech), E-cadherin (1:2000; #14,472; CST, Danvers, MA, United States), Vimentin (1:2000; #5741; CST), ZEB (1:1000; #9585; CST), or β-actin (1:4000; sc-81178; Santa Cruz Biotechnology, Dallas, TX, United States) overnight at 4°C.

Techniques: Marker, Transfection, Incubation